RNA mediated transcriptional control is an emerging principle in gene expression. X-inactivation and genomic imprinting in mammals and dosage compensation in Drosophila represent prime examples where RNAs play a crucial role in gene expression. However, the molecular mechanisms involved in these processes are poorly understood. The long-term objectives of this project are to understand the regulatory roles of such nuclear RNAs in transcriptional regulation, and to elucidate the molecular mechanisms by which these RNAs associate with chromatin and change the DNA's propensity for gene expression. The general strategy of the proposed research is to investigate RNA mediated mechanisms in gene expression using the genetically established system of Drosophila. We have recently isolated two male-specific genes, roX1 and roX2, that function as regulatory RNAs in the male nucleus. roX1 and roX2 RNA co-localize with the male-specific lethal proteins (MSLs) to the single male X- chromosome and are likely to play a role in dosage compensation, a mechanism analogous to mammalian X-inactivation. The major interest in the proposed research will be directed towards understanding the function and regulation of the roX RNAs in male Drosophila. To accomplish these goals, we propose the following four specific aims: (1) Induce and characterize mutations in roX2 and investigate genetic interactions between roX1 and roX2; (2) Determine the parameters for X-chromosome localization and define structural components assoicated with the roX RNAs; (3) Study the consequences of ectopic roX RNA expression on the regulation of linked genes in an in vivo model system; (4) Determine the regulatory mechanism underlying male-specific roX expression and define essential cis-regulatory elements. The roX genes of Drosophila provide an ideal model system to investigate the roles of RNAs in transcriptional regulation and should advance our understanding of the principles that govern RNA mediated gene regulation.